Ord, M. | bioRxiv Science

bioRxiv2021

Cdc6 is sequentially regulated by PP2A-Cdc55, Cdc14 and Sic1 for origin licensing in S. cerevisiae

Cdc6, a subunit of the pre-replicative complex, contains multiple regulatory Cdk1 consensus sites, SP or TP motifs. In S. cerevisiae, Cdk1 phosphorylates Cdc6-T7 to recruit Cks1, the Cdk1 phospho-adaptor in S-phase, for subsequent multisite phosphorylation and protein degradation. Cdc6 accumulates in mitosis and is tightly bound by Clb2 through N-terminal phosphorylation in order to prevent premature origin licensing and degradation. It has been extensively studied how Cdc6 phosphorylation is regulated by the Cyclin-Cdk1 complex. However, a detailed mechanism on how Cdc6 phosphorylation is reversed by phosphatases has not been elucidated. Here, we show that PP2ACdc55 dephosphorylates Cdc6 N-terminal sites to release Clb2. Cdc14 dephosphorylates the C-terminal phospho-degron, leading to Cdc6 stabilization in mitosis. In addition, the Cdk1 inhibitor, Sic1, releases Clb2{middle dot}Cdk1{middle dot}Cks1 from Cdc6 to load Mcm2-7 on the chromatin upon mitotic exit. Thus, pre-RC assembly and origin licensing is promoted by the attenuation of distinct CDK-dependent Cdc6 inhibitory mechanisms.

cell biology↗

bioRxiv2021

Multisite phosphorylation by Cdk1 initiates delayed negative feedback to control mitotic transcription

Cell-cycle progression is driven by the phosphorylation of cyclin-dependent kinase (Cdk) substrates1-3. The order of substrate phosphorylation depends in part on the general rise in Cdk activity during the cell cycle4-7, together with variations in substrate docking to sites on associated cyclin and Cks subunits3, 6, 8-10. Many substrates are modified at multiple sites to provide more complex regulation9, 11-14. Here, we describe an elegant regulatory circuit based on multisite phosphorylation of Ndd1, a transcriptional co-activator of genes required for mitotic progression15, 16. As cells enter mitosis, Ndd1 phosphorylation by Cdk1 is known to promote mitotic cyclin (CLB2) gene transcription, resulting in positive feedback17-20. Consistent with these findings, we show that low Cdk1 activity promotes CLB2 expression at mitotic entry. We also find, however, that CLB2 expression is inhibited by high levels of Cdk1 activity in a mitotic arrest. Inhibition is accompanied by Ndd1 degradation, and we present evidence that high mitotic Cdk1-Clb2 activity generates phosphodegrons on Ndd1, leading to its degradation. Complete Ndd1 phosphorylation by the Clb2-Cdk1-Cks1 complex requires the phosphothreonine-binding site of Cks1, as well as a novel phosphate-binding pocket on the cyclin Clb221. We therefore propose that initial phosphorylation by Cdk1 primes the protein for secondary phosphorylation at phosphodegrons, resulting in degradation only at high Cdk1 activity. Together, our results suggest that rising levels of mitotic Cdk1 activity act at multiple phosphorylation sites on Ndd1, first triggering rapid positive feedback and then promoting delayed negative feedback, resulting in a pulse of mitotic gene expression.

cell biology↗